Candida albicans is the most important fungal pathogen in the developed world, causing roughly half of the 400,000+ annual deaths attributed to candidiasis worldwide. The patients who are most at-risk for developing disseminated or invasive candidiasis are those with deficient innate immunity and we have long studied the dynamic and complex interaction between C. albicans and macrophages. Phagocytosis stimulates a dramatic program of transcriptional and metabolic changes that enable the cell to resist the stresses imposed by the macrophage. This includes a switch to a gluconeogenic growth mode in which the cell apparently utilizes a variety of nonfermentable carbon sources and we have shown that some of the pathways needed to assimilate these compounds are required for full virulence in animal models. Our data indicate that amino acids are particularly important sources of carbon in the phagosome. C. albicans uses the catabolism of amino acids to generate ammonia (derived from the amino and side chain amines) that is excreted into the extracellular space to neutralize the culture media in vitro and the phagosome in vivo. Strains unable to generate this ammonia, such as a mutant lacking Stp2, a transcription factor that regulates amino acid uptake and catabolism, occupy a more acidic phagosome and, as a result, fail to form hyphae and are more readily killed by the macrophage. We have identified a family of genes known as ATO, for Ammonia Transport Outward, that is greatly expanded in C. albicans relative to other fungi (ten homologs whereas Saccharomyces cerevisiae has three). Many, but not all, of the ten genes are induced in phagocytosed cells and an overlapping set are regulated by Stp2. We have shown that a null mutant of ATO5 or a dominant point mutant in ATO1 impairs alkalinization in vitro and in the phagosome, and renders the cell modestly more sensitive to killing by macrophages. We suggest that these phenotypes are limited because of the potential for redundancy in this large gene family, and because some of the ATO proteins are specialized for activity on other substrates. Indeed, we have identified similar alkalinization phenomena when cells are grown on N-acetylglucosamine and carboxylic acids such as ?- ketoglutarate, pyruvate and lactate and these are not affected by ato1 or ato5 mutations. We also present evidence that ATO proteins are required to maintain cytosolic pH homeostasis under weak acid stresses. Together this leads us to hypothesize that the ATO proteins are outward (that is, cytosol to extracellular space) transporters of acetate and/or ammonia that help maintain physiological cellular pH in the weak acid stress conditions like that of the phagosome. Thus, the ATOs are particularly important for the fitness of C. albicans in the host, and this is why the family is so significantly expanded. We will test this in two specific aims, the first of which is to use novel genetic technologies (the Cas9/CRISPR system) to probe redundancy and specialization in the ATO family by generating and testing multiple mutants. In a second aim, we will test the hypothesis that the ATOs are required for cellular pH homeostasis using pHluorin, a pH-sensitive GFP variant, assessing pH-dependent toxicity of organic acids, and testing whether ato-deficient strains have defects in exporting acetate or other acids. Together, these approaches will be the most detailed analysis yet of the ATO enigma - a conserved protein family that is absent in metazoans, about which virtually nothing is known beyond a potential role in host-pathogen interactions.